Nucleotide Excision Repair (NER) is a versatile DNA repair pathway involved in the removal of diverse lesions, including those formed by UV light, from DNA. The importance of NER is underscored by the fact that the cancer-prone inherited disorder xeroderma pigmentosum is caused by defects in various NER genes. NER acts by the concerted action of over 30 proteins that sequentially assemble at sites of UV damage. Although the basic biochemical transactions involved in NER have been discerned, it is less well understood how the individual steps are coordinated to ensure smooth progression through the pathway. The research outlined here combines biochemical, cell biological, chemical and structural approaches to study the regulation and coordination of the dual incision and repair synthesis steps in NER. The proposal is guided by the hypothesis that the incisions, 5'to the lesion by ERCC1-XPF and 3'to the lesion by XPG, are temporarily and spatially regulated to avoid the formation of deleterious breaks and gaps in the process. We will test the specific hypothesis that this coordination is achieved by protein-protein interactions, DNA binding and catalytic activities as follows: 1) New structural information will be used to reassign and characterize the XPA-ERCC1 interaction domains and we will study how XPA recruits ERCC1-XPF to sites of NER. 2) We will study how individual DNA binding domains of ERCC1-XPF contribute to its catalytic activity and progression through the NER pathway. 3) We will investigate how the order of incisions by ERCC1-XPF and XPG and the coordination of incision and repair synthesis activities avoids the formation of single-stranded DNA gaps following excision of a 30 nucleotide long oligonuelcotide containing the damage. Our studies will provide new insight into the molecular basis of the regulation of a complex biochemical pathway in human cells as well as the molecular basis of inherited disease and the etiology of cancer. Since NER proteins also counteract the action of many clinically important antitumor agents, our studies will also contribute new targets for therapeutic intervention. Project Narrative: Nucleotide excision repair (NER) is a complex DNA repair pathway that counteracts damage to DNA caused by UV light, environmental agents and cancer chemotherapeutic drugs. Defects in NER genes result in the cancer-prone inherited disorder xeroderma pigmentosum and our interdisciplinary studies of this pathway contribute to our understanding of the molecular basis of human disease. Since NER proteins also contribute to the resistance of tumor cells to anti-cancer agents our investigations may also provide new targets for anti- tumor therapy.